A CRISPR-dCas9 System for Assaying and Selecting for RNase III Activity In Vivo in Escherichia coli.

Ribonuclease III (RNase III) and RNase III-like ribonucleases have a wide range of important functions and are found in all organisms, yet a simple and high-throughput in vivo method for measuring RNase III activity does not exist. Typical methods for measuring RNase III activity rely on in vitro RNA analysis or in vivo methods that are not suitable for high-throughput analysis. In this study, we describe our development of a deactivated Cas9 (dCas9)-based in vivo assay for RNase III activity that utilizes RNase III's cleavage of the 5'-untranslated region (UTR) of its own messenger RNA. The key molecule in the system is a hybrid guide RNA (gRNA) between the 5'-UTR of RNase III and gGFP, a gRNA that works with dCas9 to repress GFP expression. This fusion must be cleaved by RNase III for full GFP repression. Our system uses GFP fluorescence to report on Escherichia coli RNase III activity in culture and on an individual cell basis, making it effective for selecting individual cells through fluorescence-activated cell sorting. Homology between enzymes within the RNase III family suggests this assay might be adapted to measure the activity of other enzymes in the RNase III family such as human Dicer or Drosha.

Association of Frailty, Age, and Biological Sex With Severe Acute Respiratory Syndrome Coronavirus 2 Messenger RNA Vaccine-Induced Immunity in Older Adults.

BACKGROUND: Male sex and old age are risk factors for severe coronavirus disease 2019, but the intersection of sex and aging on antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has not been characterized. METHODS: Plasma samples were collected from older adults (aged 75-98 years) before and after 3 doses of SARS-CoV-2 mRNA vaccination, and from younger adults (aged 18-74 years) post-dose 2, for comparison. Antibody binding to SARS-CoV-2 antigens (spike protein [S], S receptor-binding domain, and nucleocapsid), functional activity against S, and live-virus neutralization were measured against the vaccine virus and the Alpha, Delta, and Omicron variants of concern (VOCs). RESULTS: Vaccination induced greater antibody titers in older females than in older males, with both age and frailty associated with reduced antibody responses in males but not females. Responses declined significantly in the 6 months after the second dose. The third dose restored functional antibody responses and eliminated disparities caused by sex, age, and frailty in older adults. Responses to the VOCs, particularly the Omicron variant, were significantly reduced relative to the vaccine virus, with older males having lower titers to the VOCs than older females. Older adults had lower responses to the vaccine and VOC viruses than younger adults, with greater disparities in males than in females. CONCLUSIONS: Older and frail males may be more vulnerable to breakthrough infections owing to low antibody responses before receipt of a third vaccine dose. Promoting third dose coverage in older adults, especially males, is crucial to protecting this vulnerable population.

Adaptive immune responses in vaccinated patients with symptomatic SARS-CoV-2 Alpha infection.

Benchmarks for protective immunity from infection or severe disease after SARS-CoV-2 vaccination are still being defined. Here, we characterized virus neutralizing and ELISA antibody levels, cellular immune responses, and viral variants in 4 separate groups: healthy controls (HCs) weeks (early) or months (late) following vaccination in comparison with symptomatic patients with SARS-CoV-2 after partial or full mRNA vaccination. During the period of the study, most symptomatic breakthrough infections were caused by the SARS-CoV-2 Alpha variant. Neutralizing antibody levels in the HCs were sustained over time against the vaccine parent virus but decreased against the Alpha variant, whereas IgG titers and T cell responses against the parent virus and Alpha variant declined over time. Both partially and fully vaccinated patients with symptomatic infections had lower virus neutralizing antibody levels against the parent virus than the HCs, similar IgG antibody titers, and similar virus-specific T cell responses measured by IFN-γ. Compared with HCs, neutralization activity against the Alpha variant was lower in the partially vaccinated infected patients and tended to be lower in the fully vaccinated infected patients. In this cohort of breakthrough infections, parent virus neutralization was the superior predictor of breakthrough infections with the Alpha variant of SARS-CoV-2.

Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in E. coli.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

A redox-based electrogenetic CRISPR system to connect with and control biological information networks.

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.

Bacterial co-culture with cell signaling translator and growth controller modules for autonomously regulated culture composition.

Synthetic biology and metabolic engineering have expanded the possibilities for engineered cell-based systems. The addition of non-native biosynthetic and regulatory components can, however, overburden the reprogrammed cells. In order to avoid metabolic overload, an emerging area of focus is on engineering consortia, wherein cell subpopulations work together to carry out a desired function. This strategy requires regulation of the cell populations. Here, we design a synthetic co-culture controller consisting of cell-based signal translator and growth-controller modules that, when implemented, provide for autonomous regulation of the consortia composition. The system co-opts the orthogonal autoinducer AI-1 and AI-2 cell-cell signaling mechanisms of bacterial quorum sensing (QS) to enable cross-talk between strains and a QS signal-controlled growth rate controller to modulate relative population densities. We further develop a simple mathematical model that enables cell and system design for autonomous closed-loop control of population trajectories.

Plasmid-encoded protein attenuates Escherichia coli swimming velocity and cell growth, not reprogrammed regulatory functions.

In addition to engineering new pathways for synthesis, synthetic biologists rewire cells to carry out "programmable" functions, an example being the creation of wound-healing probiotics. Engineering regulatory circuits and synthetic machinery, however, can be deleterious to cell function, particularly if the "metabolic burden" is significant. Here, a synthetic regulatory circuit previously constructed to direct Escherichia coli to swim toward hydrogen peroxide, a signal of wound generation, was shown to work even with coexpression of antibiotic resistance genes and genes associated with lactose utilization. We found, however, that cotransformation with a second vector constitutively expressing GFP (as a marker) and additionally conferring resistance to kanamycin and tetracycline resulted in slower velocity (Δ~6 µm/s) and dramatically reduced growth rate (Δ > 50%). The additional vector did not, however, alter the run-and-tumble ratio or directional characteristics of H2 O2 -dependent motility. The main impact of this additional burden was limited to slowing cell velocity and growth, suggesting that reprogrammed cell motility by minimally altering native regulatory circuits can be maintained even when extraneous burden is placed on the host cell. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2778, 2019.

Development of Cell-Based Sentinels for Nitric Oxide: Ensuring Marker Expression and Unimodality.

We generated "sentinel" bacteria that respond to the biomarker nitric oxide (NO) and produce a homogeneous and strong fluorescent response. Our dual-plasmid system consists of a signal "relay" vector that employs an NO-responsive promoter that amplifies the native signal (via expression of T7 Polymerase (T7Pol)) to a second vector responsible for GFP expression. Importantly, to achieve an optimal "sentinel" response, we developed strategies that balance the transcriptional load within cells by altering (i) translation and (ii) activity of the T7Pol. Our optimized genetic circuitry was then used to transform commensal E. coli Nissle, as a proof-of-concept toward an ingestible cell-based sensor for Crohn's disease (CD) that, in turn, is marked by elevated levels of intestinal NO. Thus, the "biosensors" demonstrated here may serve as a simple diagnostic tool, contrasting the standard of care including colonoscopies or biopsies.

Evidence of link between quorum sensing and sugar metabolism in Escherichia coli revealed via cocrystal structures of LsrK and HPr.

Quorum sensing (QS), a bacterial process that regulates population-scale behavior, is mediated by small signaling molecules, called autoinducers (AIs), that are secreted and perceived, modulating a "collective" phenotype. Because the autoinducer AI-2 is secreted by a wide variety of bacterial species, its "perception" cues bacterial behavior. This response is mediated by the lsr (LuxS-regulated) operon that includes the AI-2 transporter LsrACDB and the kinase LsrK. We report that HPr, a phosphocarrier protein central to the sugar phosphotransferase system of Escherichia coli, copurifies with LsrK. Cocrystal structures of an LsrK/HPr complex were determined, and the effects of HPr and phosphorylated HPr on LsrK activity were assessed. LsrK activity is inhibited when bound to HPr, revealing new linkages between QS activity and sugar metabolism. These findings help shed new light on the abilities of bacteria to rapidly respond to changing nutrient levels at the population scale. They also suggest new means of manipulating QS activity among bacteria and within various niches.

Engineering bacterial motility towards hydrogen-peroxide.

Synthetic biologists construct innovative genetic/biological systems to treat environmental, energy, and health problems. Many systems employ rewired cells for non-native product synthesis, while a few have employed the rewired cells as 'smart' devices with programmable function. Building on the latter, we developed a genetic construct to control and direct bacterial motility towards hydrogen peroxide, one of the body's immune response signaling molecules. A motivation for this work is the creation of cells that can target and autonomously treat disease, the latter signaled by hydrogen peroxide release. Bacteria naturally move towards a variety of molecular cues (e.g., nutrients) in the process of chemotaxis. In this work, we engineered bacteria to recognize and move towards hydrogen peroxide, a non-native chemoattractant and potential toxin. Our system exploits oxyRS, the native oxidative stress regulon of E. coli. We first demonstrated H2O2-mediated upregulation motility regulator, CheZ. Using transwell assays, we showed a two-fold increase in net motility towards H2O2. Then, using a 2D cell tracking system, we quantified bacterial motility descriptors including velocity, % running (of tumble/run motions), and a dynamic net directionality towards the molecular cue. In CheZ mutants, we found that increased H2O2 concentration (0-200 µM) and induction time resulted in increased running speeds, ultimately reaching the native E. coli wild-type speed of ~22 µm/s with a ~45-65% ratio of running to tumbling. Finally, using a microfluidic device with stable H2O2 gradients, we characterized responses and the potential for "programmed" directionality towards H2O2 in quiescent fluids. Overall, the synthetic biology framework and tracking analysis in this work will provide a framework for investigating controlled motility of E. coli and other 'smart' probiotics for signal-directed treatment.

Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching.

This work sets out to provide a self-assembled biopolymer capsule activated with a multi-functional enzyme for localized delivery. This enzyme, SsoPox, which is a lactonase and phosphotriesterase, provides a means of interrupting bacterial communication pathways that have been shown to mediate pathogenicity. Here we demonstrate the capability to express, purify and attach SsoPox to the natural biopolymer chitosan, preserving its activity to "neutralize" long-chain autoinducer-1 (AI-1) communication molecules. Attachment is shown via non-specific binding and by engineering tyrosine and glutamine affinity 'tags' at the C-terminus for covalent linkage. Subsequent degradation of AI-1, in this case N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), serves to "quench" bacterial quorum sensing (QS), silencing intraspecies communication. By attaching enzymes to pH-responsive chitosan that, in turn, can be assembled into various forms, we demonstrate device-based flexibility for enzyme delivery. Specifically, we have assembled quorum-quenching capsules consisting of an alginate inner core and an enzyme "decorated" chitosan shell that are shown to preclude bacterial QS crosstalk, minimizing QS mediated behaviors.

Incorporating LsrK AI-2 quorum quenching capability in a functionalized biopolymer capsule.

Antibacterial resistance is an issue of increasing severity as current antibiotics are losing their effectiveness and fewer antibiotics are being developed. New methods for combating bacterial virulence are required. Modulating molecular communication among bacteria can alter phenotype, including attachment to epithelia, biofilm formation, and even toxin production. Intercepting and modulating communication networks provide a means to attenuate virulence without directly interacting with the bacteria of interest. In this work, we target communication mediated by the quorum sensing (QS) bacterial autoinducer-2, AI-2. We have assembled a capsule of biological polymers alginate and chitosan, attached an AI-2 processing kinase, LsrK, and provided substrate, ATP, for enzymatic alteration of AI-2 in culture fluids. Correspondingly, AI-2 mediated QS activity is diminished. All components of this system are "biofabricated"-they are biologically derived and their assembly is accomplished using biological means. Initially, component quantities and kinetics were tested as assembled in microtiter plates. Subsequently, the identical components and assembly means were used to create the "artificial cell" capsules. The functionalized capsules, when introduced into populations of bacteria, alter the dynamics of the AI-2 bacterial communication, attenuating QS activated phenotypes. We envision the assembly of these and other capsules or similar materials, as means to alter QS activity in a biologically compatible manner and in many environments, including in humans.

Controlling localization of Escherichia coli populations using a two-part synthetic motility circuit: An accelerator and brake.

Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as "tunable" factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as "pseudotaxis." Escherichia coli, absent the motility regulator cheZ, swim sporadically, missing the traditional "run" in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be "programmed" to swim toward the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to "brake" the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit.

Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression.

Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering.

Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX.

The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role in the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca(2+) -regulation remains unclear. It was found previously that for NCX in the absence of Ca(2+) the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca(2+) -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but also it should explain the distinctive behavior of Drosophila melanogaster's sodium/calcium exchanger, CALX, for which Ca(2+) -binding to CBD1 inhibits Ca(2+) exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca(2+) modulates CBD1 and CBD2 interdomain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca(2+) regulation of the Na(+) /Ca(2+) exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca(2+) -binding to CBD1 controlling ion transport across the plasma membrane.

Calcium binding to leptospira outer membrane antigen LipL32 is not necessary for its interaction with plasma fibronectin, collagen type IV, and plasminogen.

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca(2+). Recent crystal structures have been obtained for the protein in the apo- and Ca(2+)-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca(2+) and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca(2+) binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca(2+) affinity as the wild-type protein. We then evaluated if Ca(2+) binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca(2+) ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

Increased immunogenicity to LipL32 of Leptospira interrogans when expressed as a fusion protein with the cholera toxin B subunit.

Leptospirosis is one of the most widespread zoonosis in the world. The development of a recombinant leptospira vaccine remains a challenge. In this study, we cloned the Leptospira interrogans open reading frame (ORF) coding the external membrane protein LipL32, an immunodominant antigen found in all pathogenic leptospira, downstream of the highly immunogenic cholera toxin B subunit (CTB) ORF. Expression and assembly of the CTB-LipL32 fusion protein into oligomeric structures of pentameric size were observed in soluble fractions by Western blot analysis. The CTB-LipL32 protein demonstrated strong affinity for monosialotetrahexosylgaglioside (GM1-ganglioside) in an enzyme-linked immunosorbent assay (ELISA), suggesting that the antigenic sites for binding and proper folding of the pentameric CTB structure were conserved. Furthermore, antisera against LipL32 also recognized the CTB-LipL32 fusion protein, suggesting that LipL32 also conserved its antigenic sites, a fact confirmed by an ELISA assay showing soluble CTB-LipL32 recognition by sera from convalescent patients. In addition, soluble CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32. The data presented here provide support for CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.

Structure and calcium-binding activity of LipL32, the major surface antigen of pathogenic Leptospira sp.

Leptospirosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-A-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystallographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and laminin.

Crystallization and preliminary X-ray analysis of LipL32 from Leptospira interrogans serovar Copenhageni.

LipL32 is a major surface protein that is expressed during infection by pathogenic Leptospira. Here, the crystallization of recombinant LipL32(21-272), which corresponds to the mature LipL32 protein minus its N-terminal lipid-anchored cysteine residue, is described. Selenomethionine-labelled LipL32(21-272) crystals diffracted to 2.25 A resolution at a synchrotron source. The space group was P3(1)21 or P3(2)21 and the unit-cell parameters were a = b = 126.7, c = 96.0 A.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity.

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.